Methods for isolating a target analyte from a heterogenous sample

ABSTRACT

The invention generally relates to methods of using compositions that include sets of magnetic particles, members of each set being conjugated to an antibody specific for a pathogen, and magnets to isolate a pathogen from a body fluid sample.

RELATED APPLICATION

The present application is a continuation of U.S. non-provisionalapplication Ser. No. 14/487,692, filed Sep. 5, 2014, which is acontinuation of U.S. non-provisional application Ser. No. 13/091,510,filed Apr. 21, 2011, which claims the benefit of and priority to U.S.provisional patent application Ser. No. 61/326,588, filed Apr. 21, 2010,the contents of each of which are incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The invention generally relates to methods of using compositions thatinclude sets of magnetic particles, members of each set being conjugatedto an antibody specific for a pathogen, and magnets to isolate apathogen from a body fluid sample.

BACKGROUND

Blood-borne pathogens are a significant healthcare problem. A delayed orimproper diagnosis of a bacterial infection can result in sepsis, aserious, and often deadly, inflammatory response to the infection.Sepsis is the 10^(th) leading cause of death in the United States. Earlydetection of bacterial infections in blood is the key to preventing theonset of sepsis. Traditional methods of detection and identification ofblood-borne infection include blood culture and antibioticsusceptibility assays. Those methods typically require culturing cells,which can be expensive and can take as long as 72 hours. Often, septicshock will occur before cell culture results can be obtained.

Alternative methods for detection of pathogens, particularly bacteria,have been described by others. Those methods include molecular detectionmethods, antigen detection methods, and metabolite detection methods.Molecular detection methods, whether involving hybrid capture orpolymerase chain reaction (PCR), require high concentrations of purifiedDNA for detection. Both antigen detection and metabolite detectionmethods also require a relatively large amount of bacteria and have highlimit of detection (usually >10⁴ CFU/mL), thus requiring an enrichmentstep prior to detection. This incubation/enrichment period is intendedto allow for the growth of bacteria and an increase in bacterial cellnumbers to more readily aid in identification. In many cases, a seriesof two or three separate incubations is needed to isolate the targetbacteria. However, such enrichment steps require a significant amount oftime (e.g., at least a few days to a week) and can potentiallycompromise test sensitivity by killing some of the cells sought to bemeasured.

There is a need for methods for isolating target analytes, such asbacteria, from a sample, such as a blood sample, without an additionalenrichment step. There is also a need for methods of isolating targetanalytes that are fast and sensitive in order to provide data forpatient treatment decisions in a clinically relevant time frame.

SUMMARY

The present invention provides methods for isolating pathogens in abiological sample. The invention allows the rapid detection of pathogenat very low levels in the sample; thus enabling early and accuratedetection and identification of the pathogen. The invention is carriedout using sets of magnetic particles, members of each set beingconjugated to a binding element, such as an antibody, that is specificfor a pathogen. The invention allows detection of pathogen in aheterogeneous biological sample at levels of from about 10 CFU/ml toabout 1 CFU/ml.

Methods of the invention involve introducing magnetic particles to abiological sample (e.g., a tissue or body fluid sample). The sample isincubated to allow the particles to bind to pathogen in the sample, anda magnetic field is applied to capture pathogen/magnetic particlecomplexes on a surface. Optionally, the surface can be washed with awash solution that reduces particle aggregation, thereby isolatingpathogen/magnetic particle complexes. A particular advantage ofcompositions of the invention is for capture and isolation of bacteriaand fungi directly from blood samples at low concentrations that arepresent in many clinical samples (as low as 1 CFU/ml of bacteria in ablood sample). Preferably, the magnetic particles comprise a pathogenbinding element that has one or more magnetic particles attached to it.

In certain aspects, methods of the invention involve obtaining aheterogeneous sample including a pathogen, exposing the sample to acocktail including a plurality of sets of magnetic particles, members ofeach set being conjugated to an antibody specific for a pathogen, andseparating particle bound pathogen from other components in the sample.Methods of the invention may further involve characterizing thepathogen. Characterizing may include identifying the pathogen by anytechnique known in the art. Exemplary techniques include sequencingnucleic acid derived from the pathogen or amplifying the nucleic acid.

The antibodies conjugated to the particles may be either monoclonal orpolyclonal antibodies. Methods of the invention may be used to isolatepathogen from heterogeneous sample. In particular embodiments, theheterogeneous sample is a blood sample.

Since each set of particles is conjugated with antibodies have differentspecificities for different pathogens, compositions of the invention maybe provided such that each set of antibody conjugated particles ispresent at a concentration designed for detection of a specific pathogenin the sample. In certain embodiments, all of the sets are provided atthe same concentration. Alternatively, the sets are provided atdifferent concentrations.

To facilitate detection of the different sets of pathogen/magneticparticle complexes the particles may be differently labeled. Anydetectable label may be used with compositions of the invention, such asfluorescent labels, radiolabels, enzymatic labels, and others. Inparticular embodiments, the detectable label is an optically-detectablelabel, such as a fluorescent label. Exemplary fluorescent labels includeCy3, Cy5, Atto, cyanine, rhodamine, fluorescien, coumarin, BODIPY,alexa, and conjugated multi-dyes.

Methods of the invention may be used to isolate only gram positivebacteria from a sample. Alternatively, methods of the invention may beused to isolate only gram negative bacteria from a sample. In certainembodiments, methods of the invention are used to isolate both grampositive and gram negative bacteria from a sample. In still otherembodiments, methods isolate specific pathogen from a sample. Exemplarybacterial species that may be captured and isolated by methods of theinvention include E. coli, Listeria, Clostridium, Mycobacterium,Shigella, Borrelia, Campylobacter, Bacillus, Salmonella, Staphylococcus,Enterococcus, Pneumococcus, Streptococcus, and a combination thereof.

Methods of the invention are not limited to isolating pathogens from abody fluid. Methods of the invention may be designed to isolate othertypes of target analytes, such as fungi, protein, a cell, a virus, anucleic acid, a receptor, a ligand, or any molecule known in the art.

Compositions used in methods of the invention may use any type ofmagnetic particle. Magnetic particles generally fall into two broadcategories. The first category includes particles that are permanentlymagnetizable, or ferromagnetic; and the second category includesparticles that demonstrate bulk magnetic behavior only when subjected toa magnetic field. The latter are referred to as magnetically responsiveparticles. Materials displaying magnetically responsive behavior aresometimes described as superparamagnetic. However, materials exhibitingbulk ferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic particles. In other embodiments, the magneticparticles include at least 70% superparamagnetic particles by weight. Incertain embodiments, the superparamagnetic particles are from about 100nm to about 250 nm in diameter. In certain embodiments, the magneticparticle is an iron-containing magnetic particle. In other embodiments,the magnetic particle includes iron oxide or iron platinum.

Another aspect of the invention provides methods for isolating pathogenfrom a heterogeneous sample, that involve labeling pathogen from abiological sample with a cocktail including a plurality of sets ofmagnetic particles, members of each set being conjugated to an antibodyspecific for a pathogen, exposing the sample to a magnetic field toisolate pathogen conjugated to the particles, and isolating particlebound pathogen from other components of the sample. Methods of theinvention may further involve eluting pathogen from the particles.Methods of the invention may further involve characterizing thepathogen. Characterizing may include identifying the pathogen by anytechnique known in the art. Exemplary techniques include sequencingnucleic acid derived from the pathogen or amplifying the nucleic acid.

DETAILED DESCRIPTION

The invention generally relates to methods of using compositions thatinclude sets of magnetic particles, members of each set being conjugatedto an antibody specific for a pathogen, and magnets to isolate apathogen from a body fluid sample. Certain fundamental technologies andprinciples are associated with binding magnetic materials to targetentities and subsequently separating by use of magnet fields andgradients. Such fundamental technologies and principles are known in theart and have been previously described, such as those described inJaneway (Immunobiology, 6^(th) edition, Garland Science Publishing), thecontent of which is incorporated by reference herein in its entirety.

Composition used in methods of the invention may use any type ofmagnetic particle. Production of magnetic particles and particles foruse with the invention are known in the art. See for example Giaever(U.S. Pat. No. 3,970,518), Senyi et al. (U.S. Pat. No. 4,230,685), Dodinet al. (U.S. Pat. No. 4,677,055), Whitehead et al. (U.S. Pat. No.4,695,393), Benjamin et al. (U.S. Pat. No. 5,695,946), Giaever (U.S.Pat. No. 4,018,886), Rembaum (U.S. Pat. No. 4,267,234), Molday (U.S.Pat. No. 4,452,773), Whitehead et al. (U.S. Pat. No. 4,554,088), Forrest(U.S. Pat. No. 4,659,678), Liberti et al. (U.S. Pat. No. 5,186,827), Ownet al. (U.S. Pat. No. 4,795,698), and Liberti et al. (WO 91/02811), thecontent of each of which is incorporated by reference herein in itsentirety.

Magnetic particles generally fall into two broad categories. The firstcategory includes particles that are permanently magnetizable, orferromagnetic; and the second category includes particles thatdemonstrate bulk magnetic behavior only when subjected to a magneticfield. The latter are referred to as magnetically responsive particles.Materials displaying magnetically responsive behavior are sometimesdescribed as superparamagnetic. However, materials exhibiting bulkferromagnetic properties, e.g., magnetic iron oxide, may becharacterized as superparamagnetic when provided in crystals of about 30nm or less in diameter. Larger crystals of ferromagnetic materials, bycontrast, retain permanent magnet characteristics after exposure to amagnetic field and tend to aggregate thereafter due to strongparticle-particle interaction. In certain embodiments, the particles aresuperparamagnetic particles. In certain embodiments, the magneticparticle is an iron containing magnetic particle. In other embodiments,the magnetic particle includes iron oxide or iron platinum.

In certain embodiments, the magnetic particles include at least about10% superparamagnetic particles by weight, at least about 20%superparamagnetic particles by weight, at least about 30%superparamagnetic particles by weight, at least about 40%superparamagnetic particles by weight, at least about 50%superparamagnetic particles by weight, at least about 60%superparamagnetic particles by weight, at least about 70%superparamagnetic particles by weight, at least about 80%superparamagnetic particles by weight, at least about 90%superparamagnetic particles by weight, at least about 95%superparamagnetic particles by weight, or at least about 99%superparamagnetic particles by weight. In a particular embodiment, themagnetic particles include at least about 70% superparamagneticparticles by weight.

In certain embodiments, the superparamagnetic particles are less than100 nm in diameter. In other embodiments, the superparamagneticparticles are about 150 nm in diameter, are about 200 nm in diameter,are about 250 nm in diameter, are about 300 nm in diameter, are about350 nm in diameter, are about 400 nm in diameter, are about 500 nm indiameter, or are about 1000 nm in diameter. In a particular embodiment,the superparamagnetic particles are from about 100 nm to about 250 nm indiameter.

In certain embodiments, the particles are particles (e.g.,nanoparticles) that incorporate magnetic materials, or magneticmaterials that have been functionalized, or other configurations as areknown in the art. In certain embodiments, nanoparticles may be used thatinclude a polymer material that incorporates magnetic material(s), suchas nanometal material(s). When those nanometal material(s) orcrystal(s), such as Fe₃O₄, are superparamagnetic, they may provideadvantageous properties, such as being capable of being magnetized by anexternal magnetic field, and demagnetized when the external magneticfield has been removed. This may be advantageous for facilitating sampletransport into and away from an area where the sample is being processedwithout undue particle aggregation.

One or more or many different nanometal(s) may be employed, such asFe₃O₄, FePt, or Fe, in a core-shell configuration to provide stability,and/or various others as may be known in the art. In many applications,it may be advantageous to have a nanometal having as high a saturatedmoment per volume as possible, as this may maximize gradient relatedforces, and/or may enhance a signal associated with the presence of theparticles. It may also be advantageous to have the volumetric loading ina particle be as high as possible, for the same or similar reason(s). Inorder to maximize the moment provided by a magnetizable nanometal, acertain saturation field may be provided. For example, for Fe₃O₄superparamagnetic particles, this field may be on the order of about0.3T.

The size of the nanometal containing particle may be optimized for aparticular application, for example, maximizing moment loaded upon atarget, maximizing the number of particles on a target with anacceptable detectability, maximizing desired force-induced motion,and/or maximizing the difference in attached moment between the labeledtarget and non-specifically bound targets or particle aggregates orindividual particles. While maximizing is referenced by example above,other optimizations or alterations are contemplated, such as minimizingor otherwise desirably affecting conditions.

In an exemplary embodiment, a polymer particle containing 80 wt % Fe₃O₄superparamagnetic particles, or for example, 90 wt % or highersuperparamagnetic particles, is produced by encapsulatingsuperparamagnetic particles with a polymer coating to produce a particlehaving a diameter of about 250 nm.

Each set of magnetic particles has a target-specific binding moiety thatallows for each set to specifically bind the target of interest in theheterogeneous sample. The target-specific moiety may be any moleculeknown in the art and will depend on the target to be captured andisolated. Exemplary target-specific binding moieties include nucleicacids, proteins, ligands, antibodies, aptamers, and receptors.

In particular embodiments, the target-specific binding moiety is anantibody, such as an antibody that binds a particular bacterium. Generalmethodologies for antibody production, including criteria to beconsidered when choosing an animal for the production of antisera, aredescribed in Harlow et al. (Antibodies, Cold Spring Harbor Laboratory,pp. 93-117, 1988). For example, an animal of suitable size such asgoats, dogs, sheep, mice, or camels are immunized by administration ofan amount of immunogen, such the target bacteria, effective to producean immune response. An exemplary protocol is as follows. The animal isinjected with 100 milligrams of antigen resuspended in adjuvant, forexample Freund's complete adjuvant, dependent on the size of the animal,followed three weeks later with a subcutaneous injection of 100micrograms to 100 milligrams of immunogen with adjuvant dependent on thesize of the animal, for example Freund's incomplete adjuvant. Additionalsubcutaneous or intraperitoneal injections every two weeks withadjuvant, for example Freund's incomplete adjuvant, are administereduntil a suitable titer of antibody in the animal's blood is achieved.Exemplary titers include a titer of at least about 1:5000 or a titer of1:100,000 or more, i.e., the dilution having a detectable activity. Theantibodies are purified, for example, by affinity purification oncolumns containing protein G resin or target-specific affinity resin.

The technique of in vitro immunization of human lymphocytes is used togenerate monoclonal antibodies. Techniques for in vitro immunization ofhuman lymphocytes are well known to those skilled in the art. See, e.g.,Inai, et al., Histochemistry, 99(5):335 362, May 1993; Mulder, et al.,Hum. Immunol., 36(3):186 192, 1993; Harada, et al., J. Oral Pathol.Med., 22(4):145 152, 1993; Stauber, et al., J. Immunol. Methods,161(2):157 168, 1993; and Venkateswaran, et al., Hybridoma, 11(6) 729739, 1992. These techniques can be used to produce antigen-reactivemonoclonal antibodies, including antigen-specific IgG, and IgMmonoclonal antibodies.

Any antibody or fragment thereof having affinity and specific for thebacteria of interest is within the scope of the invention providedherein. Immunomagnetic particles against Salmonella are provided inVermunt et al. (J. Appl. Bact. 72:112, 1992). Immunomagnetic particlesagainst Staphylococcus aureus are provided in Johne et al. (J. Clin.Microbiol. 27:1631, 1989). Immunomagnetic particles against Listeria areprovided in Skjerve et al. (Appl. Env. Microbiol. 56:3478, 1990).Immunomagnetic particles against Escherichia coli are provided in Lundet al. (J. Clin. Microbiol. 29:2259, 1991).

Methods for attaching the target-specific binding moiety to the magneticparticle are known in the art. Coating magnetic particles withantibodies is well known in the art, see for example Harlow et al.(Antibodies, Cold Spring Harbor Laboratory, 1988), Hunter et al.(Immunoassays for Clinical Chemistry, pp. 147-162, eds., ChurchillLivingston, Edinborough, 1983), and Stanley (Essentials in Immunologyand Serology, Delmar, pp. 152-153, 2002). Such methodology can easily bemodified by one of skill in the art to bind other types oftarget-specific binding moieties to the magnetic particles. Certaintypes of magnetic particles coated with a functional moiety arecommercially available from Sigma-Aldrich (St. Louis, Mo.).

Since each set of particles is conjugated with antibodies havingdifferent specificities for different pathogens, compositions used inmethods of the invention may be provided such that each set of antibodyconjugated particles is present at a concentration designed fordetection of a specific pathogen in the sample. In certain embodiments,all of the sets are provided at the same concentration. Alternatively,the sets are provided at different concentrations. For example,compositions may be designed such that sets that bind gram positivebacteria are added to the sample at a concentration of 2×10⁹ particlesper/ml, while sets that bind gram negative bacteria are added to thesample at a concentration of 4×10⁹ particles per/ml. Compositions usedwith methods of the invention are not affected by antibodycross-reactivity. However, in certain embodiments, sets are specificallydesigned such that there is no cross-reactivity between differentantibodies and different sets.

Methods of the invention may be used to isolate only gram positivebacteria from a sample. Alternatively, methods of the invention may beused to isolate only gram negative bacteria from a sample. In certainembodiments, methods of the invention are used to isolate both grampositive and gram negative bacteria from a sample. Such compositionsallow for isolation of essentially all bacteria from a sample.

In still other embodiments, compositions used with methods of theinvention are designed to isolate specific pathogen from a sample.Exemplary bacterial species that may be captured and isolated by methodsof the invention include E. coli, Listeria, Clostridium, Mycobacterium,Shigella, Borrelia, Campylobacter, Bacillus, Salmonella, Staphylococcus,Enterococcus, Pneumococcus, Streptococcus, and a combination thereof.These sets can be mixed together to isolate for example, E. coli andListeria; or E. coli, Listeria, and Clostridium; or Mycobacterium,Campylobacter, Bacillus, Salmonella, and Staphylococcus, etc. Anycombination of sets may be used and compositions of the invention willvary depending on the suspected pathogen or pathogens to be isolated.

Capture of a wide range of target microorganisms simultaneously can beachieved by utilizing antibodies specific to target class, such aspan-Gram-positive antibodies, pan-Gran-negative antibodies or antibodiesspecific to a subset of organisms of a certain class. Further, expandedreactivity can be achieved by mixing particles of different reactivity.It was shown in our experiments that addition of high concentration ofnon-specific particles does not interfere with the capture efficiency oftarget-specific particles. Similarly, several different particlepreparations can be combined to allow for the efficient capture ofdesired pathogens. In certain embodiments the particles can be utilizedat a concentration between 1×10⁸ and 5×10¹⁰ particles/mL.

In certain embodiments the expanded coverage can be provided by mixingantibodies with different specificity before attaching them to magneticparticles. Purified antibodies can be mixed and conjugated to activatedmagnetic particle using standard methods known in the art.

To facilitate detection of the different sets of pathogen/magneticparticle complexes the particles may be differently labeled. Anydetectable label may be used with compositions of the invention, such asfluorescent labels, radiolabels, enzymatic labels, and others. Thedetectable label may be directly or indirectly detectable. In certainembodiments, the exact label may be selected based, at least in part, onthe particular type of detection method used. Exemplary detectionmethods include radioactive detection, optical absorbance detection,e.g., UV-visible absorbance detection, optical emission detection, e.g.,fluorescence; phosphorescence or chemiluminescence; Raman scattering.Preferred labels include optically-detectable labels, such asfluorescent labels. Examples of fluorescent labels include, but are notlimited to, 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid;acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Atto dyes, Cy3; Cy5; Cy5.5; Cy7; IRD 700;IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.Preferred fluorescent labels are cyanine-3 and cyanine-5. Labels otherthan fluorescent labels are contemplated by the invention, includingother optically-detectable labels. Methods of linking fluorescent labelsto magnetic particles or antibodies are known in the art.

Methods of the invention may be used to isolate pathogen from anyheterogeneous sample. In particular embodiments, methods of theinvention isolate a pathogen from body fluid. A body fluid refers to aliquid material derived from, for example, a human or other mammal. Suchbody fluids include, but are not limited to, mucus, blood, plasma,serum, serum derivatives, bile, phlegm, saliva, sweat, amniotic fluid,mammary fluid, urine, sputum, and cerebrospinal fluid (CSF), such aslumbar or ventricular CSF. A body fluid may also be a fine needleaspirate. A body fluid also may be media containing cells or biologicalmaterial.

In particular embodiments, the fluid is blood. Methods of the inventionallow for bacteria in a blood sample to be isolated and detected at alevel as low as or even lower than 1 CFU/ml. Blood may be collected in acontainer, such as a blood collection tube (e.g., VACUTAINER, test tubespecifically designed for venipuncture, commercially available fromBecton, Dickinson and company). In certain embodiments, a solution isadded that prevents or reduces aggregation of endogenous aggregatingfactors, such as heparin in the case of blood.

The blood sample is then mixed with compositions as described above togenerate a mixture that is allowed to incubate such that thecompositions bind to at least one bacterium in the blood sample. Thetype or types of bacteria that will bind compositions of the inventionwill depend on the design of the composition, i.e., which antibodyconjugated particles are used. The mixture is allowed to incubate for asufficient time to allow for the composition to bind to the pathogen inthe blood. The process of binding the composition to the pathogenassociates a magnetic moment with the pathogen, and thus allows thepathogen to be manipulated through forces generated by magnetic fieldsupon the attached magnetic moment.

In general, incubation time will depend on the desired degree of bindingbetween the pathogen and the compositions of the invention (e.g., theamount of moment that would be desirably attached to the pathogen), theamount of moment per target, the amount of time of mixing, the type ofmixing, the reagents present to promote the binding and the bindingchemistry system that is being employed. Incubation time can be anywherefrom about 5 seconds to a few days. Exemplary incubation times rangefrom about 10 seconds to about 2 hours. Binding occurs over a wide rangeof temperatures, generally between 15° C. and 40° C.

In certain embodiments, a buffer solution is added to the sample alongwith the compositions of the invention. An exemplary buffer includesTris(hydroximethyl)-aminomethane hydrochloride at a concentration ofabout 75 mM. It has been found that the buffer composition, mixingparameters (speed, type of mixing, such as rotation, shaking etc., andtemperature) influence binding. It is important to maintain osmolalityof the final solution (e.g., blood+buffer) to maintain high labelefficiency. In certain embodiments, buffers used in methods of theinvention are designed to prevent lysis of blood cells, facilitateefficient binding of targets with magnetic particles and to reduceformation of particle aggregates. It has been found that the buffersolution containing 300 mM NaCl, 75 mM Tris-HCl pH 8.0 and 0.1% Tween 20meets these design goals.

Without being limited by any particular theory or mechanism of action,it is believed that sodium chloride is mainly responsible formaintaining osmolality of the solution and for the reduction ofnon-specific binding of magnetic particle through ionic interaction.Tris(hydroximethyl)-aminomethane hydrochloride is a well establishedbuffer compound frequently used in biology to maintain pH of a solution.It has been found that 75 mM concentration is beneficial and sufficientfor high binding efficiency. Likewise, Tween 20 is widely used as a milddetergent to decrease nonspecific attachment due to hydrophobicinteractions. Various assays use Tween 20 at concentrations ranging from0.01% to 1%. The 0.1% concentration appears to be optimal for theefficient labeling of bacteria, while maintaining blood cells intact.

Additional compounds can be used to modulate the capture efficiency byblocking or reducing non-specific interaction with blood components andeither magnetic particles or pathogens. For example, chelatingcompounds, such as EDTA or EGTA, can be used to prevent or minimizeinteractions that are sensitive to the presence of Ca²⁺ or Mg²⁺ ions.

An alternative approach to achieve high binding efficiency whilereducing time required for the binding step is to use static mixer, orother mixing devices that provide efficient mixing of viscous samples athigh flow rates, such as at or around 5 mL/min. In one embodiment, thesample is mixed with binding buffer in ratio of, or about, 1:1, using amixing interface connector. The diluted sample then flows through amixing interface connector where it is mixed with target-specificnanoparticles. Additional mixing interface connectors providing mixingof sample and antigen-specific nanoparticles can be attached downstreamto improve binding efficiency. The combined flow rate of the labeledsample is selected such that it is compatible with downstreamprocessing.

After binding of the compositions to the pathogen in the sample to formpathogen/magnetic particle complexes, a magnetic field is applied to themixture to capture the complexes on a surface. Components of the mixturethat are not bound to magnetic particles will not be affected by themagnetic field and will remain free in the mixture. Methods andapparatuses for separating target/magnetic particle complexes from othercomponents of a mixture are known in the art. For example, a steel meshmay be coupled to a magnet, a linear channel or channels may beconfigured with adjacent magnets, or quadrapole magnets with annularflow may be used. Other methods and apparatuses for separatingtarget/magnetic particle complexes from other components of a mixtureare shown in Rao et al. (U.S. Pat. No. 6,551,843), Liberti et al. (U.S.Pat. No. 5,622,831), Hatch et al. (U.S. Pat. No. 6,514,415), Benjamin etal. (U.S. Pat. No. 5,695,946), Liberti et al. (U.S. Pat. No. 5,186,827),Wang et al. (U.S. Pat. No. 5,541,072), Liberti et al. (U.S. Pat. No.5,466,574), and Terstappen et al. (U.S. Pat. No. 6,623,983), the contentof each of which is incorporated by reference herein in its entirety.

In certain embodiments, the magnetic capture is achieved at highefficiency by utilizing a flow-through capture cell with a number ofstrong rare earth bar magnets placed perpendicular to the flow of thesample. When using a flow chamber with flow path cross-section 0.5 mm×20mm (h×w) and 7 bar NdFeB magnets, the flow rate could be as high as 5mL/min or more, while achieving capture efficiency close to 100%.

The above described type of magnetic separation produces efficientcapture of a target analyte and the removal of a majority of theremaining components of a sample mixture. However, such a processproduces a sample that contains a very high percent of magneticparticles that are not bound to target analytes because the magneticparticles are typically added in excess, as well as non-specific targetentities. Non-specific target entities may for example be bound at amuch lower efficiency, for example 1% of the surface area, while atarget of interest might be loaded at 50% or nearly 100% of theavailable surface area or available antigenic cites. However, even 1%loading may be sufficient to impart force necessary for trapping in amagnetic gradient flow cell or sample chamber.

For example, in the case of immunomagnetic binding of bacteria or fungiin a blood sample, the sample may include: bound targets at aconcentration of about 1/mL or a concentration less than about 10⁶/mL;background particles at a concentration of about 10⁷/ml to about10¹⁰/ml; and non-specific targets at a concentration of about 10/ml toabout 10⁵/ml.

The presence of magnetic particles that are not bound to target analytesand non-specific target entities on the surface that includes thetarget/magnetic particle complexes interferes with the ability tosuccessfully detect the target of interest. The magnetic capture of theresulting mix, and close contact of magnetic particles with each otherand bound targets, result in the formation of aggregate that is hard todispense and which might be resistant or inadequate for subsequentprocessing or analysis steps. In order to remove magnetic particles thatare not bound to target analytes and non-specific target entities, thesurface may be washed with a wash solution that reduces particleaggregation, thereby isolating target/magnetic particle complexes fromthe magnetic particles that are not bound to target analytes andnon-specific target entities. The wash solution minimizes the formationof the aggregates.

Any wash solution that imparts a net negative charge to the magneticparticle that is not sufficient to disrupt interaction between thetarget-specific moiety of the magnetic particle and the target analytemay be used. Without being limited by any particular theory or mechanismof action, it is believed that attachment of the negatively chargedmolecules in the wash solution to magnetic particles provides netnegative charge to the particles and facilitates dispersal ofnon-specifically aggregated particles. At the same time, the netnegative charge is not sufficient to disrupt strong interaction betweenthe target-specific moiety of the magnetic particle and the targetanalyte (e.g., an antibody-antigen interaction). Exemplary solutionsinclude heparin, Tris-HCl, Tris-borate-EDTA (TBE), Tris-acetate-EDTA(TAE), Tris-cacodylate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid), PBS (phosphatebuffered saline), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid), MES(2-N-morpholino)ethanesulfonic acid), Tricine(N-(Tri(hydroximethyl)methyl)glycine), and similar buffering agents. Incertain embodiments, only a single wash cycle is performed. In otherembodiments, more than one wash cycle is performed.

In particular embodiments, the wash solution includes heparin. Forembodiments in which the body fluid sample is blood, the heparin alsoreduces probability of clotting of blood components after magneticcapture. The bound targets are washed with heparin-containing buffer 1-3times to remove blood components and to reduce formation of aggregates.

Once the target/magnetic particle complexes are isolated, the target maybe analyzed by a multitude of existing technologies, such as miniatureNMR, Polymerase Chain Reaction (PCR), mass spectrometry, fluorescentlabeling and visualization using microscopic observation, fluorescent insitu hybridization (FISH), growth-based antibiotic sensitivity tests,and variety of other methods that may be conducted with purified targetwithout significant contamination from other sample components. In oneembodiment, isolated bacteria are eluted from the magnetic particles andare lysed with a chaotropic solution, and DNA is bound to DNA extractionresin. After washing of the resin, the bacterial DNA is eluted and usedin quantitative RT-PCR to detect the presence of a specific species,and/or, subclasses of bacteria.

In another embodiment, captured bacteria is removed from the magneticparticles to which they are bound and the processed sample is mixed withfluorescent labeled antibodies specific to the bacteria or fluorescentGram stain. After incubation, the reaction mixture is filtered through0.2 μm to 1.0 μm filter to capture labeled bacteria while allowingmajority of free particles and fluorescent labels to pass through thefilter. Bacteria is visualized on the filter using microscopictechniques, e.g. direct microscopic observation, laser scanning or otherautomated methods of image capture. The presence of bacteria is detectedthrough image analysis. After the positive detection by visualtechniques, the bacteria can be further characterized using PCR orgenomic methods.

Detection of bacteria of interest can be performed by use of nucleicacid probes following procedures which are known in the art. Suitableprocedures for detection of bacteria using nucleic acid probes aredescribed, for example, in Stackebrandt et al. (U.S. Pat. No.5,089,386), King et al. (WO 90/08841), Foster et al. (WO 92/15883), andCossart et al. (WO 89/06699), each of which is hereby incorporated byreference.

A suitable nucleic acid probe assay generally includes sample treatmentand lysis, hybridization with selected probe(s), hybrid capture, anddetection. Lysis of the bacteria is necessary to release the nucleicacid for the probes. The nucleic acid target molecules are released bytreatment with any of a number of lysis agents, including alkali (suchas NaOH), guanidine salts (such as guanidine thiocyanate), enzymes (suchas lysozyme, mutanolysin and proteinase K), and detergents. Lysis of thebacteria, therefore, releases both DNA and RNA, particularly ribosomalRNA and chromosomal DNA both of which can be utilized as the targetmolecules with appropriate selection of a suitable probe. Use of rRNA asthe target molecule(s), may be advantageous because rRNAs constitute asignificant component of cellular mass, thereby providing an abundanceof target molecules. The use of rRNA probes also enhances specificityfor the bacteria of interest, that is, positive detection withoutundesirable cross-reactivity which can lead to false positives or falsedetection.

Hybridization includes addition of the specific nucleic acid probes. Ingeneral, hybridization is the procedure by which two partially orcompletely complementary nucleic acids are combined, under definedreaction conditions, in an anti-parallel fashion to form specific andstable hydrogen bonds. The selection or stringency of thehybridization/reaction conditions is defined by the length and basecomposition of the probe/target duplex, as well as by the level andgeometry of mis-pairing between the two nucleic acid strands. Stringencyis also governed by such reaction parameters as temperature, types andconcentrations of denaturing agents present and the type andconcentration of ionic species present in the hybridization solution.

The hybridization phase of the nucleic acid probe assay is performedwith a single selected probe or with a combination of two, three or moreprobes. Probes are selected having sequences which are homologous tounique nucleic acid sequences of the target organism. In general, afirst capture probe is utilized to capture formed hybrid molecules. Thehybrid molecule is then detected by use of antibody reaction or by useof a second detector probe which may be labelled with a radioisotope(such as phosphorus-32) or a fluorescent label (such as fluorescein) orchemiluminescent label.

Detection of bacteria of interest can also be performed by use of PCRtechniques. A suitable PCR technique is described, for example, inVerhoef et al. (WO 92/08805). Such protocols may be applied directly tothe bacteria captured on the magnetic particles. The bacteria iscombined with a lysis buffer and collected nucleic acid target moleculesare then utilized as the template for the PCR reaction.

For detection of the selected bacteria by use of antibodies, isolatedbacteria are contacted with antibodies specific to the bacteria ofinterest. As noted above, either polyclonal or monoclonal antibodies canbe utilized, but in either case have affinity for the particularbacteria to be detected. These antibodies, will adhere/bind to materialfrom the specific target bacteria. With respect to labeling of theantibodies, these are labeled either directly or indirectly with labelsused in other known immunoassays. Direct labels may include fluorescent,chemiluminescent, bioluminescent, radioactive, metallic, biotin orenzymatic molecules. Methods of combining these labels to antibodies orother macromolecules are well known to those in the art. Examplesinclude the methods of Hijmans, W. et al. (1969), Clin. Exp. Immunol. 4,457-, for fluorescein isothiocyanate, the method of Goding, J. W.(1976), J. Immunol. Meth. 13, 215-, for tetramethylrhodamineisothiocyanate, and the method of Ingrall, E. (1980), Meth. in Enzymol.70, 419-439 for enzymes.

These detector antibodies may also be labeled indirectly. In this casethe actual detection molecule is attached to a secondary antibody orother molecule with binding affinity for the anti-bacteria cell surfaceantibody. If a secondary antibody is used it is preferably a generalantibody to a class of antibody (IgG and IgM) from the animal speciesused to raise the anti-bacteria cell surface antibodies. For example,the second antibody may be conjugated to an enzyme, either alkalinephosphatase or to peroxidase. To detect the label, after the bacteria ofinterest is contacted with the second antibody and washed, the isolatedcomponent of the sample is immersed in a solution containing achromogenic substrate for either alkaline phosphatase or peroxidase. Achromogenic substrate is a compound that can be cleaved by an enzyme toresult in the production of some type of detectable signal which onlyappears when the substrate is cleaved from the base molecule. Thechromogenic substrate is colorless, until it reacts with the enzyme, atwhich time an intensely colored product is made. Thus, material from thebacteria colonies adhered to the membrane sheet will become an intenseblue/purple/black color, or brown/red while material from other colonieswill remain colorless. Examples of detection molecules includefluorescent substances, such as 4-methylumbelliferyl phosphate, andchromogenic substances, such as 4-nitrophenylphosphate,3,3′,5,5′-tetramethylbenzidine and2,2′-azino-di-[3-ethelbenz-thiazoliane sulfonate (6)]. In addition toalkaline phosphatase and peroxidase, other useful enzymes includeβ-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,α-mannosidase, galactose oxidase, glucose oxidase and hexokinase.

Detection of bacteria of interest using NMR may be accomplished asfollows. In the use of NMR as a detection methodology, in which a sampleis delivered to a detector coil centered in a magnet, the target ofinterest, such as a magnetically labeled bacterium, may be delivered bya fluid medium, such as a fluid substantially composed of water. In sucha case, the magnetically labeled target may go from a region of very lowmagnetic field to a region of high magnetic field, for example, a fieldproduced by an about 1 to about 2 Tesla magnet. In this manner, thesample may traverse a magnetic gradient, on the way into the magnet andon the way out of the magnet. As may be seen via equations 1 and 2below, the target may experience a force pulling into the magnet in thedirection of sample flow on the way into the magnet, and a force intothe magnet in the opposite direction of flow on the way out of themagnet. The target may experience a retaining force trapping the targetin the magnet if flow is not sufficient to overcome the gradient force.m dot (del B)=F   Equation 1v _(t) =−F/(6*p*n*r)   Equation 2where n is the viscosity, r is the particle diameter, F is the vectorforce, B is the vector field, and m is the vector moment of the particle

Magnetic fields on a path into a magnet may be non-uniform in thetransverse direction with respect to the flow into the magnet. As such,there may be a transverse force that pulls targets to the side of acontainer or a conduit that provides the sample flow into the magnet.Generally, the time it takes a target to reach the wall of a conduit isassociated with the terminal velocity and is lower with increasingviscosity. The terminal velocity is associated with the drag force,which may be indicative of creep flow in certain cases. In general, itmay be advantageous to have a high viscosity to provide a higher dragforce such that a target will tend to be carried with the fluid flowthrough the magnet without being trapped in the magnet or against theconduit walls.

Newtonian fluids have a flow characteristic in a conduit, such as around pipe, for example, that is parabolic, such that the flow velocityis zero at the wall, and maximal at the center, and having a paraboliccharacteristic with radius. The velocity decreases in a direction towardthe walls, and it is easier to magnetically trap targets near the walls,either with transverse gradients force on the target toward the conduitwall, or in longitudinal gradients sufficient to prevent target flow inthe pipe at any position. In order to provide favorable fluid drag forceto keep the samples from being trapped in the conduit, it may beadvantageous to have a plug flow condition, wherein the fluid velocityis substantially uniform as a function of radial position in theconduit.

When NMR detection is employed in connection with a flowing sample, thedetection may be based on a perturbation of the NMR water signal causedby a magnetically labeled target (Sillerud et al., JMR (Journal ofMagnetic Resonance), vol. 181, 2006). In such a case, the sample may beexcited at time 0, and after some delay, such as about 50 ms or about100 ms, an acceptable measurement (based on a detected NMR signal) maybe produced. Alternatively, such a measurement may be producedimmediately after excitation, with the detection continuing for someduration, such as about 50 ms or about 100 ms. It may be advantageous todetect the NMR signal for substantially longer time durations after theexcitation.

By way of example, the detection of the NMR signal may continue for aperiod of about 2 seconds in order to record spectral information athigh-resolution. In the case of parabolic or Newtonian flow, theperturbation excited at time 0 is typically smeared because the wateraround the perturbation source travels at different velocity, dependingon radial position in the conduit. In addition, spectral information maybe lost due to the smearing or mixing effects of the differential motionof the sample fluid during signal detection. When carrying out an NMRdetection application involving a flowing fluid sample, it may beadvantageous to provide plug-like sample flow to facilitate desirableNMR contrast and/or desirable NMR signal detection.

Differential motion within a flowing Newtonian fluid may havedeleterious effects in certain situations, such as a situation in whichspatially localized NMR detection is desired, as in magnetic resonanceimaging. In one example, a magnetic object, such as a magneticallylabeled bacterium, is flowed through the NMR detector and its presenceand location are detected using MRI techniques. The detection may bepossible due to the magnetic field of the magnetic object, since thisfield perturbs the magnetic field of the fluid in the vicinity of themagnetic object. The detection of the magnetic object is improved if thefluid near the object remains near the object. Under these conditions,the magnetic perturbation may be allowed to act longer on any givenvolume element of the fluid, and the volume elements of the fluid soaffected will remain in close spatial proximity. Such a stronger, morelocalized magnetic perturbation will be more readily detected using NMRor MRI techniques.

If a Newtonian fluid is used to carry the magnetic objects through thedetector, the velocity of the fluid volume elements will depend onradial position in the fluid conduit. In such a case, the fluid near amagnetic object will not remain near the magnetic object as the objectflows through the detector. The effect of the magnetic perturbation ofthe object on the surrounding fluid may be smeared out in space, and thestrength of the perturbation on any one fluid volume element may bereduced because that element does not stay within range of theperturbation. The weaker, less-well-localized perturbation in the samplefluid may be undetectable using NMR or MRI techniques.

Certain liquids, or mixtures of liquids, exhibit non-parabolic flowprofiles in circular conduits. Such fluids may exhibit non-Newtonianflow profiles in other conduit shapes. The use of such a fluid may proveadvantageous as the detection fluid in an application employing anNMR-based detection device. Any such advantageous effect may beattributable to high viscosity of the fluid, a plug-like flow profileassociated with the fluid, and/or other characteristic(s) attributed tothe fluid that facilitate detection. As an example, a shear-thinningfluid of high viscosity may exhibit a flow velocity profile that issubstantially uniform across the central regions of the conduitcross-section. The velocity profile of such a fluid may transition to azero or very low value near or at the walls of the conduit, and thistransition region may be confined to a very thin layer near the wall.

Not all fluids, or all fluid mixtures, are compatible with the NMRdetection methodology. In one example, a mixture of glycerol and watercan provide high viscosity, but the NMR measurement is degraded becauseseparate NMR signals are detected from the water and glycerol moleculesmaking up the mixture. This can undermine the sensitivity of the NMRdetector. In another example, the non-water component of the fluidmixture can be chosen to have no NMR signal, which may be achieved byusing a perdeuterated fluid component, for example, or using aperfluorinated fluid component. This approach may suffer from the lossof signal intensity since a portion of the fluid in the detection coildoes not produce a signal.

Another approach may be to use a secondary fluid component thatconstitutes only a small fraction of the total fluid mixture. Such alow-concentration secondary fluid component can produce an NMR signalthat is of negligible intensity when compared to the signal from themain component of the fluid, which may be water. It may be advantageousto use a low-concentration secondary fluid component that does notproduce an NMR signal in the detector. For example, a perfluorinated orperdeuterated secondary fluid component may be used. The fluid mixtureused in the NMR detector may include one, two, or more than twosecondary components in addition to the main fluid component. The fluidcomponents employed may act in concert to produce the desired fluid flowcharacteristics, such as high-viscosity and/or plug flow. The fluidcomponents may be useful for providing fluid characteristics that areadvantageous for the performance of the NMR detector, for example byproviding NMR relaxation times that allow faster operation or highersignal intensities.

A non-Newtonian fluid may provide additional advantages for thedetection of objects by NMR or MRI techniques. As one example, theobjects being detected may all have substantially the same velocity asthey go through the detection coil. This characteristic velocity mayallow simpler or more robust algorithms for the analysis of thedetection data. As another example, the objects being detected may havefixed, known, and uniform velocity. This may prove advantageous indevices where the position of the detected object at later times isneeded, such as in a device that has a sequestration chamber orsecondary detection chamber down-stream from the NMR or MRI detectioncoil, for example.

In an exemplary embodiment, sample delivery into and out of a 1.7Tcylindrical magnet using a fluid delivery medium containing 0.1% to 0.5%Xanthan gum in water was successfully achieved. Such delivery issuitable to provide substantially plug-like flow, high viscosity, suchas from about 10 cP to about 3000 cP, and good NMR contrast in relationto water. Xanthan gum acts as a non-Newtonian fluid, havingcharacteristics of a non-Newtonian fluid that are well know in the art,and does not compromise NMR signal characteristics desirable for gooddetection in a desirable mode of operation.

In certain embodiments, methods of the invention are useful for directdetection of bacteria from blood. Such a process is described here.Sample is collected in sodium heparin tube by venipuncture, acceptablesample volume is about 1 mL to 10 mL. Sample is diluted with bindingbuffer and superparamagnetic particles having target-specific bindingmoieties are added to the sample, followed by incubation on a shakingincubator at 37° C. for about 30 min to 120 min. Alternative mixingmethods can also be used. In a particular embodiment, sample is pumpedthrough a static mixer, such that reaction buffer and magnetic particlesare added to the sample as the sample is pumped through the mixer. Thisprocess allows for efficient integration of all components into a singlefluidic part, avoids moving parts and separate incubation vessels andreduces incubation time.

Capture of the labeled targets allows for the removal of bloodcomponents and reduction of sample volume from 30 mL to 5 mL. Thecapture is performed in a variety of magnet/flow configurations. Incertain embodiments, methods include capture in a sample tube on ashaking platform or capture in a flow-through device at flow rate of 5mL/min, resulting in total capture time of 6 min.

After capture, the sample is washed with wash buffer including heparinto remove blood components and free particles. The composition of thewash buffer is optimized to reduce aggregation of free particles, whilemaintaining the integrity of the particle/target complexes.

The detection method is based on a miniature NMR detector tuned to themagnetic resonance of water. When the sample is magnetically homogenous(no bound targets), the NMR signal from water is clearly detectable andstrong. The presence of magnetic material in the detector coil disturbsthe magnetic field, resulting in reduction in water signal. One of theprimary benefits of this detection method is that there is no magneticbackground in biological samples which significantly reduces therequirements for stringency of sample processing. In addition, since thedetected signal is generated by water, there is a built-in signalamplification which allows for the detection of a single labeledbacterium.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Sample

Blood samples from healthy volunteers were spiked with clinicallyrelevant concentrations of bacteria (1-10 CFU/mL) including bothlaboratory strains and clinical isolates of the bacterial species mostfrequently found in bloodstream infections.

Example 2 Antibody Preparation

In order to generate polyclonal, pan-Gram-positive bacteria-specificIgG, a goat was immunized by first administering bacterial antigenssuspended in complete Freund's adjuvant intra lymph node, followed bysubcutaneous injection of bacterial antigens in incomplete Freund'sadjuvant in 2 week intervals. The antigens were prepared for antibodyproduction by growing bacteria to exponential phase (OD₆₀₀=0.4-0.8).Following harvest of the bacteria by centrifugation, the bacteria wasinactivated using formalin fixation in 4% formaldehyde for 4 hr at 37°C. After 3 washes of bacteria with PBS (15 min wash, centrifugation for20 min at 4000 rpm) the antigen concentration was measured using BCAassay and the antigen was used at 1 mg/mL for immunization. In order togenerate Gram-positive bacteria-specific IgG, several bacterial specieswere used for inoculation: Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium and Enterococcus fecalis.

The immune serum was purified using affinity chromatography on a proteinG sepharose column (GE Healthcare), and reactivity was determined usingELISA. Antibodies cross-reacting with Gram-negative bacteria and fungiwere removed by absorption of purified IgG with formalin-fixedGram-negative bacteria and fungi. The formalin-fixed organisms wereprepared similar to as described above and mixed with IgG. Afterincubation for 2 hrs at room temperature, the preparation wascentrifuged to remove bacteria. Final antibody preparation was clarifiedby centrifugation and used for the preparation of antigen-specificmagnetic particles.

Pan-Gram-negative IgG were generated in a similar fashion usinginactivated Enterobacter cloacae, Pseudomonas aeruginosa, Serratiamarcescens and other gram-negative bacteria as immunogens. The IgGfraction of serum was purified using protein-G affinity chromatographyas described above.

Similarly, target specific antibodies were generated by inoculation ofgoats using formalin-fixed bacteria, immunization was performed with 2or more closely related organisms.

Example 3 Preparation of Antigen-Specific Magnetic Particles

Superparamagnetic particles were synthesized by encapsulating iron oxidenanoparticles (5-15 nm diameter) in a latex core and labeling with goatIgG. Ferrofluid containing nanoparticles in organic solvent wasprecipitated with ethanol, nanoparticles were resuspended in aqueoussolution of styrene and surfactant Hitenol BC-10, and emulsified usingsonication. The mixture was allowed to equilibrate overnight withstirring and filtered through 1.2 and 0.45 μm filters to achieve uniformmicelle size. Styrene, acrylic acid and divinylbenzene were added incarbonate buffer at pH 9.6. The polymerization was initiated in amixture at 70° C. with the addition of K₂ 5 ₂O₈ and the reaction wasallowed to complete overnight. The synthesized particles were washed 3times with 0.1% SDS using magnetic capture, filtered through 1.2, 0.8,and 0.45 μm filters and used for antibody conjugation.

The production of particles resulted in a distribution of sizes that maybe characterized by an average size and a standard deviation. In thecase of labeling and extracting of bacteria from blood, the average sizefor optimal performance was found to be between 100 and 350 nm, forexample between 200 nm to 250 nm.

The purified IgG were conjugated to prepared particles using standardEDC/sulfo-NHS chemistry. After conjugation, the particles wereresuspended in 0.1% BSA which is used to block non-specific bindingsites on the particle and to increase the stability of particlepreparation.

Example 4 Labeling of Rare Cells Using Excess of Magnetic Nanoparticles

Bacteria, present in blood during blood-stream infection, weremagnetically labeled using the superparamagnetic particles prepared inExample 3 above. The spiked samples as described in Example 1 werediluted 3-fold with a Tris-based binding buffer and target-specificparticles, followed by incubation on a shaking platform at 37° C. for upto 2 hr. The optimal concentration of particles was determined bytitration and was found to be in the range between 1×10⁸ and 5×10¹⁰particle/mL. After incubation, the labeled targets were magneticallyseparated followed by a wash step designed to remove blood products. Seeexample 5 below.

Example 5 Magnetic Capture of Bound Bacteria

Blood including the magnetically labeled target bacteria and excess freeparticles were injected into a flow-through capture cell with a numberof strong rare earth bar magnets placed perpendicular to the flow of thesample. With using a flow chamber with flow path cross-section 0.5 mm×20mm (h×w) and 7 bar NdFeB magnets, a flow rate as high as 5 mL/min wasachieved. After flowing the mixture through the channel in the presenceof the magnet, a wash solution including heparin was flowed through thechannel. The bound targets were washed with heparin-containing bufferone time to remove blood components and to reduce formation of magneticparticle aggregates. In order to effectively wash bound targets, themagnet was removed and captured magnetic material was resuspended inwash buffer, followed by re-application of the magnetic field andcapture of the magnetic material in the same flow-through capture cell.

Removal of the captured labeled targets was possible after movingmagnets away from the capture chamber and eluting with flow of buffersolution.

What is claimed is:
 1. A method for isolating pathogen from aheterogeneous sample, the method comprising: providing a vessel thatcontains a heterogeneous sample comprising pathogen, the vessel beingcoupled to a fluidic device that comprises a channel and a magnet;introducing to the sample in the vessel a cocktail comprising aplurality of magnetic particles, conjugated to target-specific bindingmoieties that bind said pathogen, thereby forming a mixture comprisingpathogen/magnetic particle complexes; flowing the mixture from thevessel into the fluidic device and through a channel in the fluidicdevice such that said complexes are held in place by the magnet; andseparating antibody-bound pathogen being held by the magnet from othercomponents in the sample.
 2. The method of claim 1, further comprisingthe step of characterizing the pathogen.
 3. The method of claim 2,wherein said characterizing step comprises identifying the pathogen. 4.The method of claim 3, wherein said identifying step is selected fromsequencing nucleic acid derived from the pathogen and amplifying thenucleic acid.
 5. The method of claim 1, wherein the sample is a bloodsample.
 6. The method of claim 1, wherein the particles aredifferentially labeled.
 7. The method of claim 6, wherein the particlescomprise is an optical label.
 8. The method of claim 7, wherein theoptical label is a fluorescent label.
 9. The method of claim 1, whereinthe target-specific binding moieties comprise antibodies.
 10. The methodof claim 1, wherein the antibodies are monoclonal antibodies.
 11. Themethod of claim 1, wherein the antibodies are polyclonal antibodies. 12.The method of claim 1, wherein the pathogen is gram positive bacteria.13. The method of claim 1, wherein the pathogen is gram negativebacteria.
 14. The method of claim 1, wherein the pathogen is a virus.15. The method of claim 1, wherein the magnetic particles comprise aplurality of sets, wherein members of different sets are conjugated todifferent antibodies that are specific for different pathogen.
 16. Themethod of claim 15, wherein the different sets are provided at differentconcentrations.
 17. The method of claim 15, wherein the different setsare provided at the same concentrations.
 18. The method of claim 1,wherein the magnetic particles are beads.
 19. The method of claim 18,wherein the bead are from about 100 nm to about 250 nm in diameter.